TITLE
METHOD OF IDENTIFYING COMPOUNDS THAT MODULATE
INTERACTION OF ANDROGEN RECEPTOR WITH p-CATENIN
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority from U.S. Provisional Application No.
60/682,580, filed May 19, 2005, which is incorporated by reference herein
in its entirety.
FIELD OF THE INVENTION
The present invention relates to an assay for identification of
compounds that modulate the androgen-dependent interaction between
androgen receptor (AR) and p-catenin. This invention particularly relates
to the identification of molecules which may be able to disrupt the
interaction of androgen receptor and p-catenin and thereby specifically
remove the effect of p-catenin on AR signaling or remove the effect of AR
on p-catenin and Wnt signaling.
BACKGROUND OF INVENTION
Traditionally, nuclear/steroid receptor binding ligands are identified
by screening for the ability of test compounds to affect the transcription of
genes containing consensus nuclear receptor DNA elements responsive to
that nuclear/steroid receptor. However, some steroid receptors also affect,
and are affected by, non-steroidal signaling pathways in addition to their
classical steroidal transcriptional control mechanisms. In many instances,
it would be useful to have a means to identify specific compounds that are
selectively effective in modulating only the non-steroidal signaling pathway
associated with such receptor or compounds that are selectively effective
in modulating only the transcriptional activities of the receptor on genes
traditionally responsive to such receptor. Compounds with such selectivity
would have potential pharmaceutical utility in situations where modulation
of the non-steroidal pathway is desired but inhibition of classical steroid
receptor mediated transcription is not desired, or vice versa.

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It is known, for example, that androgen receptor (AR), a classic
steroid receptor which is known to activate transcription of AR-responsive
genes, also affects the Wnt signaling pathway via the androgen-mediated
interaction of AR with β-catenin (1,2,3). The Wnt pathway plays an
important role in the differentiation and functional activity of a variety of
tissues including bone, intestine, skin, and hair follicles. Alterations in the
Wnt pathway have been implicated in disease states such as osteoporosis
and prostate and colon cancer. Other conditions where Wnt signaling may
become altered include insulin resistance in polycystic ovary syndrome
and androgenic aiopecia (4,5). In many of these conditions, androgens
and AR have been implicated as being potential modulators of the Wnt
pathway.
This invention describes a novel screening assay to identify
compounds that modulate the interaction of androgen receptor with β-
catenin. When used in conjunction with standard assays measuring
modulation of classical androgen mediated AR-dependent transcription,
the assay of the invention enables the user to identify new classes of AR
modulators that selectively inhibit the ability of AR to interact with β-catenin
and modulate its activity without affecting classical AR agonist or
antagonist activity such as, for example, androgen-mediated transcription
by AR. Compounds with this selective activity could not be detected using
classical AR transcriptional assays alone.
SUMMARY OF THE INVENTION
This invention provides a method of determining if a test compound
is able to modulate the interaction between androgen receptor (AR) and β-
catenin comprising the steps of:
(a) providing a cell comprising:
(i) a DNA sequence encoding a hybrid protein
comprising a DNA binding domain fused to the NH3-
terminal region of β-catenin,
(ii) a DNA sequence comprising an upstream activation
sequence corresponding to said DNA binding domain

3
operably linked to and controlling transcription of a
reporter gene, and
(iii) a DNA sequence encoding androgen receptor protein,
(b) introducing the test compound to the ceil, optionally in the
presence of androgen; and
(c) measuring the expression of the reporter gene,
wherein a decrease or increase of expression by the reporter gene
indicates that the test molecule is able to modulate the interaction between
androgen receptor and p-catenin.
In a preferred mode, this invention further provides a method
wherein the DNA binding domain of (i) comprises a GAL-4 DNA binding
domain; and the upstream activation DNA sequence operably linked to a
reporter gene of (ii) comprises GAL-4 UAS operably linked to the reporter
gene.
In a more preferred mode, this invention further provides wherein
the NH3-terminal region p-catenin of (i) comprises amino acids 2 through
424 of human p-catenin; wherein the reporter gene is luciferase; wherein
there are multiple copies of the GAL-4 UAS sequence operably linked to
the luciferase gene; wherein the modulation accomplished by the test
compound is a decrease in the expression of the luciferase gene; and
wherein the androgen at step (b) is DHT.
Another aspect of the invention is for a method of determining if a
test compound is able to modulate the interaction between androgen
receptor and p-catenin comprising the steps of:
(a) providing a cell comprising:
(i) a DNA sequence encoding a hybrid protein
comprising a DNA binding domain fused to p-catenin,
(ii) a DNA sequence comprising an upstream activation
sequence corresponding to said DNA binding domain
operably linked to a reporter gene, and
(iii) a DNA sequence encoding androgen receptor protein,
(b) introducing the test compound to the cell, optionally in the
presence of androgen; and

4
(c) measuring the expression of the reporter gene,
wherein an increase or decrease of expression by the reporter gene
indicates that the test molecule is able to modulate the interaction between
androgen receptor and β-catenin.
Another aspect is for a method of determining if a test compound
selectively modulates the p-catenin-Wnt signaling pathway over an
androgen receptor signaling pathway comprising:
(a) identifying a test compound which increases or decreases
the expression of a gene by inhibiting the AR mediated
interaction with p-catenin, wherein the test compound
removes androgen-liganded AR repression on Wnt signaling
without repressing androgen-AR mediated transcription; and
(b) assaying the test compound of (a) to determine whether the
test compound increases or decreases the expression of a
gene through a P-catenin independent androgen receptor
signaling pathway;
whereby the test compound of (a) selectively modulates the p-catenin-Wnt
signaling pathway by inhibiting the ability androgen-liganded AR to interact
with p-catenin if the test compound fails to increase or decrease the
expression of a gene through an androgen receptor signaling pathway.
A further aspect is for a method of determining if a test compound
selectively modulates an androgen receptor signaling pathway over a p-
catenin-Wnt signaling pathway comprising:
(a) identifying a test compound which increases or decreases
the expression of a gene through an androgen receptor
signaling pathway; and
(b) assaying the test compound of (a) to determine whether the
test compound increases or decreases the ability of
androgen-liganded AR or non-liganded AR to inhibit β-
catenin/Wnt signaling;
whereby the test compound of (a) selectively modulates an androgen
receptor signaling pathway without removing androgen-liganded AR
repression of Wnt signaling or does not promote the interaction between

5
AR and β-catenin in the absence of an AR agonist resulting in the test
compound having no activity in increasing or decreasing the expression of
a gene regulated by β-catenin-Wnt signaling pathway.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 depicts the plasmid constructs used in one embodiment of the
invention.
Figure 2 is a Western Blot demonstrating that dihydrotestosterone (DHT)
stimulates the interaction between AR and p-catenin in L929 cells.
Figure 3 is a bar graph illustrating that the method of the invention
measures the DHT dependent interaction between AR and β-catenin.
Figure 4 is a bar graph illustrating that the protein-protein interaction
between p-catenin and nuclear receptors is specific for AR.
Figure 5 is a graph illustrating that the DHT stimulated interaction between
AR and p-catenin is inhibited by the AR antagonist cyproterone acetate.
Figure 6 depicts the amino acid sequence for human p-catenin
(GenBank® Accession #2208332A; SEQ ID NO:1).
DETAILED DESCRIPTION
Applicants specifically incorporate the entire contents of all cited
references in this disclosure. Further, when an amount, concentration, or
other value or parameter is given as either a range, preferred range, or a
list of upper preferable values and lower preferable values, this is to be
understood as specifically disclosing all ranges formed from any pair of
any upper range limit or preferred value and any lower range limit or
preferred value, regardless of whether ranges are separately disclosed.
Where a range of numerical values is recited herein, unless otherwise
stated, the range is intended to include the endpoints thereof, and all
integers and fractions within the range. It is not intended that the scope of
the invention be limited to the specific values recited when defining a
range.
This invention assesses the androgen dependent interaction
between AR and p-catenin, and utilizes i) a first DNA sequence comprising

6
DNA encoding a hybrid protein comprising a DNA binding domain fused to
p-catenin, ii) a second DNA sequence comprising an upstream activation
sequence able to recognize the DNA binding domain of (i) which is
operably linked to a reporter gene; and iii) a third DNA sequence encoding
AR protein. The method of the invention entails providing test compounds
to a cell comprising and able to express the DNA sequences i, ii and iii,
optionally in the presence of an androgen, to determine if the test
compound is able to modulate the androgen-stimulated interaction of p-
catenin and androgen receptor, as measured by detections of expression
of the reporter gene. If expression of the reporter gene is unaffected by
addition of the test compound to the cell, such compound is unable to
modulate the androgen-dependent interaction of p-catenin with androgen
receptor. In a preferred mode, the DNA binding domain of (i) comprises
the DNA binding domain of GAL4; the upstream activation sequence
operably linked to the reporter gene of (ii) is GAL-4-UAS; and the reporter
gene is luciferase. In a most preferred embodiment of the invention,
multiple copies of the upstream activation sequence are operably linked to
the reporter gene, and the p-catenin of (i) that is fused to the DNA binding
domain comprises amino acids 2 to 424 of human p-catenin.
Applicants have demonstrated that their assay is a selective
measure of the androgen-dependent interaction between AR and p-
catenin. Generally, in screening mode, cells transformed with the DNA
sequences of the invention are treated with an androgen such as
dihydrotestosterone (DHT) which causes the interaction of AR and p-
catenin resulting in increased reporter activity. Molecules that lower
reporter activity can be identified and further tested in secondary assays
for their ability to disrupt the interaction between AR and DNA response
consensus elements which bind liganded AR and cause activation or
inhibition of transcription. Molecules identified by this methodology in this
screen may be particularly useful as treatments for androgenic alopecia,
prostate cancer, and insulin sensitivity in polycystic ovary syndrome.
Applicants' assay is similar to the two-hybrid binding assay of Fields
et al. (6,7), which utilizes one hybrid comprising a protein fused to a DNA

7
binding domain, and a second hybrid comprising a protein fused to a
transcription activating domain. The current one-hybrid binding assay is
distinct, however, in that it should favor the identification of molecules that
may be able to disrupt the interaction of AR and P-catenin and thereby
specifically remove the effect of β-catenin on AR signaling or remove the
effect of AR on p-catenin and Wnt signaling. Further, in a preferred mode,
only a portion of the p-catenin coding sequence is fused to the GAL4 DNA
binding domain (GAL4 DBD). Because the present assay utilizes full
length, wild type AR, the entire AR protein is available for targeting and
test compounds are not blocked from binding to AR by a fusion construct.
In contrast, in the two-hybrid assay (6,7), the use of two recombinant
fusion protein constructs has the disadvantage that the natural
conformation of the target protein may be altered in the fusion construct.
An assay of the invention may be conducted in, for example, the
well-characterized and widely used CV-1 cells (African green monkey
kidney cell line) or COS cells (African green monkey kidney cell line), but
one of ordinary skill in the art would recognize that other cell lines are
suitable as well.
A further aspect of the invention is for a method of determining if a
test compound selectively modulates a p-catenin-Wnt signaling pathway
over an androgen receptor signaling pathway. In the method, test
compounds are identified based on their ability to increase or decrease the
expression of a gene by modulating the androgen-AR mediated repression
of the p-catenin-Wnt signaling pathway. "Androgen liganded androgen
receptor-mediated modulation of the p-catenin-Wnt signaling pathway" or,
as used herein, refers to a signaling pathway resulting in a transcriptional
increase or decrease of any gene modulated by the Wnt-mediated
signaling pathway initiated through an androgen receptor/p-catenin
interaction (see, e.g., Mulholland DJ eta/., J. Biol. Chem. 277:17933-43
(2002); Yang F. etal., J. Biol. Chem. 277:11336-44 (2002); Song L-N et
a/., Mol. Cell. Biol. 23:1674-87 (2003)).
Methods of assessing the interaction of androgen receptor with p-
catenin are preferentially utilized to identify test compounds capable of
increasing or decreasing the expression of a gene by modulating the

8
interaction of AR and P-catenin resulting in repression or inhibition of
androgen-AR mediated repression on the P-catenin-Wnt signaling
pathway. In a cell type specific context, the interaction between AR and p-
catenin may have a positive effect on Wnt signaling or AR mediated
signaling. In this embodiment, positive regulators of the AR-P-catenin
interaction would be developed as Wnt activators.
A test compound that positively or negatively affects the ability of
androgen-liganded AR to modulate p-catenin-Wnt transcriptional signaling
is then assayed to determine whether the test compound increases or
decreases the expression of a gene through an androgen receptor
signaling pathway. "Androgen receptor signaling pathway" or "AR
signaling pathway", as used herein, refers to the traditional transcriptional
pathway of the androgen receptor. In response to a ligand binding,
androgen receptor migrates to the nucleus of a cell where it forms a
homodimer. Upon binding to an androgen response element (ARE) as a
homodimer, agonist-bound AR stimulates transcription by recruiting a
large enzymatic co-activator complex that includes GRIP1/TIF2,
CBP/p300, and other coactivators. In addition, ligand-bound AR can also
suppress transcription via protein-protein interaction with transcription
factor complexes such as, for example, AP1, NF-KB, and Ets family.
One of ordinary skill in the art would recognize that any assay of AR
signaling pathway assessment is useful in the present invention.
Test compounds that fail to increase or decrease the expression of
a gene through an androgen signaling pathway, in the presence or
absence of natural endogenous or exogenous androgens, selectively
modulate a p-catenin-Wnt signaling pathway. By "fails to increase or
decrease the expression of a gene" is meant that no increase or decrease
of gene expression is observable through assaying techniques known to
one of ordinary skill in the art such as, for example, Northern Blotting,
Western Blotting, Southern Blotting, plasmid reporter assays, or
polymerase chain reaction (PCR), or by observing overall changes in in
vivo (animal) organ system morphology or functions known to be
modulated by androgens or p-catenin. An example of known animal
model effects of androgens would be the effects of AR modulators and

9
Wnt modulators on prostate growth/weight in rodents/mammals where AR
agonists increase prostate cell growth and organ weight and AR
antagonists inhibit this effect of androgens.
An alternate embodiment is for a method determining if a test
compound selectively modulates an androgen receptor signaling pathway
over a p-catenin-Wnt signaling pathway. In the method, test compounds
are identified based on their ability to increase or decrease the expression
of a gene through an androgen receptor signaling pathway by methods as
are well known to those of ordinary skill in the art. A test compound that
positively or negatively affects AR signaling is then assayed to determine
whether the test compound increases or decreases the expression of a
gene through an AR-mediated (3-catenin-Wnt signaling pathway as
described above.
Within the context of Applicants' disclosure, terms will have their
customary technical meaning in the art unless otherwise stated. Some
terms and aspects of the invention are further described below.
The term "androgen receptor" or "AR" refers to the AR protein as
defined by its conserved amino acid coding sequence in an active or
native structural conformation.
"Hybridization" includes a reaction in which one or more
polynucleotides react to form a complex that is stabilized via hydrogen
bonding between the bases of the nucleotide residues. The hydrogen
bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in
any other sequence-specific manner. The complex may comprise two
strands forming a duplex structure, three or more strands forming a multi-
stranded complex, a single self-hybridizing strand, or any combination of
these. A hybridization reaction may constitute a step in a more extensive
process, such as the initiation of a PCR reaction, or the enzymatic
cleavage of a polynucleotide by a ribozyme.
Hybridization reactions can be performed under conditions of
different "stringency". The stringency of a hybridization reaction includes
the difficulty with which any two nucleic acid molecules will hybridize to
one another. Under stringent conditions, nucleic acid molecules at least
65%, 70%, 75% or more identical to each other remain hybridized to each

10
other, whereas molecules with low percent identity cannot remain
hybridized. A preferred, non-limiting example of highly stringent
hybridization conditions are hybridization in 6x sodium chloride/sodium
citrate (SSC) at about 45 °C, followed by one or more washes in 0.2x
SSC, 0.1% SDS at 50 °C, preferably at 55 °C, more preferably at 60 °C,
and even more preferably at 65 °C.
When hybridization occurs in an antiparallei configuration between
two single-stranded polynucleotides, the reaction is called "annealing" and
those polynucleotides are described as "complementary". A double-
stranded polynucleotide can be "complementary" or "homologous" to
another Dolvnucleotide if hvbridization can occur between one of the
strands of the first polynucleotide and the second. "Complementarity" or
homology is quantifiable in terms of the proportion of bases in opposing
strands that are expected to hydrogen bond with each other, according to
generally accepted base-pairing rules.
The term "P-catenin" is used to encompass full-length proteins
comprising, for example, in the human protein, 781 amino acids and also
fragments of the p-catenin amino acid sequence as disclosed, for
example, in Figure 6 (SEQ ID NO:1). Preferred forms of the protein
include particularly amino acids about 1 through about 423. In other
embodiments, a (3-catenin protein has at least 65%, at least 70% amino
acid identity, more preferably 80% amino acid identity, more preferably
90%, and even more preferably, 95% amino acid identity with the amino
acid sequence shown in SEQ ID NO:1 or a portion thereof.
In another embodiment, the term "P-catenin" is used to encompass
full-length proteins or fragments thereof encoded by polynucleotides which
hybridize under stringent conditions to a polynucleotide encoding the
amino acid sequence of SEQ ID NO:1, or a fragment or complement
thereof. Preferably, the conditions are such that sequences at least 65%,
preferably at least about 70%, more preferably at least about 80%, even
more preferably at least about 85% or 90% homologous to each other
typically remain hybridized to each other. Preferably, a p-catenin
polynucleotide that hybridizes under stringent conditions to a

11
polynucleotide sequence which encodes the amino acid sequence of SEQ
ID NO:1 or fragments or complements thereof corresponds to a naturally-
occurring nucleic acid molecule.
In addition to naturally-occurring allelic variants of (3-catenin
sequences that may exist in the population, the skilled artisan will further
appreciate that minor changes may be introduced by mutation into
polynucleotide sequences which encode, for example, the amino acid
sequence of SEQ ID NO.i, thereby leading to changes in the amino acid
sequence of the encoded protein, without altering the functional activity of
a p-catenin protein. For example, nucleotide substitutions ieading to
amino acid substitutions at "non-essential" amino acid residues may be
made in a polynucleotide sequence which encodes the amino acid
sequence of SEQ ID NO:1. A "non-essential" amino acid residue is a
residue that can be altered from the wild-type sequence of a p-catenin
polynucleotide (e.g., a polynucleotide encoding the amino acid sequence
of SEQ ID NO:1) without altering the functional activity of a |3-catenin
molecule. Exemplary residues which are non-essential and, therefore,
amenable to substitution can be identified by one of ordinary skill in the art
by performing an amino acid alignment of p-catenin-related molecules and
determining residues that are not conserved. Such residues, because
they have not been conserved, are more likely amenable to substitution.
Accordingly, the term "p-catenin" also pertains to polynucleotides
encoding p-catenin proteins that contain changes in amino acid residues
that are not essential for a p-catenin activity. Such p-catenin proteins
differ in amino acid sequence of SEQ ID NO:1 yet retain an inherent p-
catenin activity. An isolated polynucleotide encoding a non-natural variant
of a p-catenin protein can be created by introducing one or more
nucleotide substitutions, additions, or deletions into a polynucleotide
sequence encoding the amino acid sequence of SEQ ID HO:1 such that
one or more amino acid substitutions, additions, or deletions are
introduced into the encoded protein. Mutations can be introduced into
SEQ ID NO:1 by standard techniques, such as site-directed mutagenesis
and PCR-mediated mutagenesis. Preferably, conservative amino acid
substitutions are made at one or more non-essential amino acid residues.

12
A "conservative amino acid substitution" is one in which the amino acid
residue is replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have been
defined in the art, including basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged
polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched
side chains (e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential
amino acid residue in a p-catenin polypeptide is preferably replaced with
another amino acid residue from the same side chain family.
Alternatively, in another embodiment, mutations can be introduced
randomly along all or part of a p-catenin coding sequence, such as by
saturation mutagenesis.
The term "NH3-terminal region of p-catenin" comprises any
contiguous amino acid sequence from amino acid one through the
armadillo repeat regions of p-catenin capable of interacting with androgen
receptor. Thus, the NH3-terminal region can comprise, for example, amino
acid 1 through amino acid 424, amino acid 2 through amino acid 424,
amino acid 3 through amino acid 424, amino acid 1 through amino acid
423, amino acid 2 through 423, amino acid 3 through 423, and so forth.
The NH3-terminal region preferably comprises armadillo repeats 1-6 of P-
catenin, more preferably armadillo repeats 1-7 of (3-catenin, and even
more preferably armadillo repeats 1-12 of (3-catenin. In another preferred
embodiment, the NH3-terminal region is amino acids 2-424 of human p-
catenin. The NH3-termina! region can comprise amino acid sequences
from only the armadillo repeat region. Embodiments comprising an NH3-
terminal region amino acid sequence contiguous with at least a portion of
the C-terminal region of p-catenin are also contemplated so long as the C-
termina! trans-activation domain of p-catenin is inactive. Conservative
substitutions, deletions, or insertions of amino acids are also contemplated
so long as an interaction between p-catenin and androgen receptor is
maintained.

13
Typical substitutions include, for example, substitution of an amino
acid with an amino acid having similar charge, hydrophobic, or
stereochemical characteristics. For example, a "conservative amino acid
substitution" may involve a substitution of a native amino acid residue with
a nonnative residue such that there is little or no effect on the polarity or
charge of the amino add residue at that position. Desired amino acid
substitutions (whether conservative or non-conservative) can be
determined by those skilled in the art at the time such substitutions are
desired. For example, amino acid substitutions can be used to identify
important residues of the molecule sequence, or to increase or decrease
the affinity of the molecules described herein. In certain embodiments,
conservative amino acid substitutions also encompass non-naturally
occurring amino acid residues which are typically incorporated by chemical
peptide synthesis rather than by synthesis in biological systems.
The term "interacting with androgen receptor" means the protein-
protein interaction between the ligand binding domain of AR and the
armadillo regions of p-catenin. The interaction between these two proteins
is caused by changes in the AR protein secondary/tertiary conformation
which is stimulated by androgen binding to AR.
The term "modulate" encompasses either a decrease or an
increase in activity; for example, a test compound can be considered to
modulate the interaction between androgen receptor and {3-caterrin if the
presence of such test compounds in the assay of the invention results in
either a decrease or increase in the expression of luciferase gene.
The term "DNA binding domain" describes any protein binding
domain that has a conserved DNA binding motif that binds in a sequence
specific manner to its conserved upstream activation sequence also
referred to as a "DNA response element" that contains the specific
nucleotide sequence or "recognition sequence" that is recognized by the
protein DNA binding domain. The DNA response element is placed in a
reporter plasmid so that proteins that bind to the DNA response element
are capable of bringing transcriptional activators in close proximity to the

14
reporter through protein-protein interactions resulting in activation of
reporter transcription.
The term "upstream activation sequence" or "UAS" includes any
DNA sequence which is able to bind the DNA binding domain which has
been selected for use in the assay as a fusion protein with (3-catenin.
The term "reporter gene" is used in the manner commonly known in
the art to describe any genetic coding sequence which is able to express a
protein or amino acid sequence that can be detected and quantitated.
Examples of well known reporter gene productions that could be used in
the assay of the invention inciude, for exampie, the enzymes iuciferase,
chloramphenicol actyltransferase, and p-galactosidase. Those skilled in
the art will know many other suitable reporter genes.
The term "test.compound" includes compounds with known
chemical structure but not necessarily with a known function or biological
activity. Test compounds could also have unidentified structures or be
mixtures of unknown compounds, for example from crude biological
samples such as plant extracts. Large numbers of compounds could be
randomly screened from "chemical libraries" which refers to collections of
purified chemical compounds or collections of crude extracts from various
sources. The chemical libraries may contain compounds that were
chemically synthesized or purified from natural products. The compounds
may comprise inorganic or organic small molecules or larger organic
compounds such as, for example, proteins, peptides, glycoproteins,
steroids, lipids, phospholipids, nucleic acids, and lipoproteins. The amount
of compound tested can very depending on the chemical library, but, for
purified (homogeneous) compound libraries, 10 uM is typically the highest
initial dose tested.
Methods of introducing test compounds to cells are well known in
the art.
The term "androgen" includes all known compounds with
androgenic activity. Androgenic activity of compounds may be determined
in a variety of ways including in cell-based AR transcription assays and in
biological activity assays where a compound can be demonstrated to have
activity that is similar to the activity of known androgens. These assays

15
can be performed using animals or tissues. For example, compounds with
androgen activity in the prostate are able to stimulate prostate growth in
rodents. Natural androgen metabolites that have biological activity can be
used and include, for example, testosterone, androstenedione,
androstanedione, and dihydrotestosterone (DHT), with DHT particularly
preferred.
The assay is tolerant of a wide concentration range of androgens.
In a preferred mode, the DHT dose is 1 nM to screen compounds.
Between 0.1 and 10 nM of androgen could be used as an initial dose to
optimize the assay in a ceii iine.
In the absence of androgen, an assay of the present invention can
also be used to identify compounds that stimulate the interaction between
AR and {3-catenin. For example, a compound with activity similar to DHT
would activate reporter activity through the stimulation of the interaction
between AR and (3-catenin.
The term "operably linked" means that a nucleic acid molecule, i.e.,
DNA, and one or more regulatory sequences (e.g., a promoter or portion
thereof) are connected in such a way as to permit transcription of mRNA
from the nucleic acid molecule or permit expression of the product (i.e., a
polypeptide) of the nucleic acid molecule when the appropriate molecules
are bound to the regulatory sequences.
The term "expression construct" means any double-stranded DNA
or double-stranded RNA designed to transcribe an RNA, e.g., a construct
that contains at lease one promoter operably linked to a downstream gene
or coding region of interest (e.g., a cDNA or genomic DNA fragment that
encodes a protein, or any RNA of interest). Transfection or transformation
of the expression construct into a recipient cell allows the cell to express
RNA or protein encoded by the expression construct. An expression
construct may be a genetically engineered plasmid, virus, or an artificial
chromosome derived from, for example, a bacteriophage, adenovirus,
retrovirus, poxvirus, or herpesvirus, or further embodiments described
under "expression vector" below. An expression construct can be
replicated in a living cell, or it can be made synthetically. For purposes of
this application, the terms "expression construct", "expression vector",

16
"vector"', and "plasmid" are used interchangeably to demonstrate the
application of the invention in a general, illustrative sense, and are not
intended to limit the invention to a particular type of expression construct.
Further, the term expression construct or vector is intended to also include
instances wherein the cell utilized for the assay already endogenously
comprises such DNA sequence.
As used herein, the terms "polynucleotide" and "oligonucleotide" are
used interchangeably, and include polymeric forms of nucleotides of any
length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
Polynucleotides may have any three-dimensional structure, and may
perform any function, known or unknown. The following are non-limiting
examples of polynucleotides: a gene or gene fragment, exons, introns,
messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes,
cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any sequence,
nucleic acid probes, and primers. A polynucleotide may comprise
modified nucleotides, such as methylated nucleotides and nucleotide
analogs. If present, modifications to the nucleotide structure may be
imparted before or after assembly of the polymer. The sequence of
nucleotides may be interrupted by non-nucleotide components. A
polynucleotide may be further modified after polymerization, such as by
conjugation with a labeling component. The term also includes both
double- and single-stranded molecules. Unless otherwise specified or
required, any embodiment of this invention that is a polynucleotide
encompasses both the double-stranded form and each of two
complementary single-stranded forms known or predicted to make up the
double-stranded form.
A polynucleotide is composed of a specific sequence of four
nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and
uracil (U) for guanine when the polynucleotide is RNA. Thus, the term
"polynucleotide sequence" is the alphabetical representation of a
polynucleotide molecule.
A "gene" includes a polynucleotide containing at least one open
reading frame that is capable of encoding a particular polypeptide or

17
protein after being transcribed and translated. Any of the polynucleotide
sequences described herein may be used to identify larger fragments or
full-length coding sequences of the gene with which they are associated.
Methods of isolating larger fragment sequences are known to those of skill
in the art, some of which are described herein.
As used herein, "expression" includes the process by which
polynucleotides are transcribed into mRNA and translated Into peptides,
polypeptides, or proteins. If the polynucleotide is derived from genomic
DNA, expression may include splicing of the mRNA, if an appropriate
eukaryotic host is selected. Regulatory elements required for expression
include promoter sequences to bind RNA polymerase and transcription
initiation sequences for ribosome binding. For example, a bacterial
expression vector includes a promoter such as the lac promoter and for
transcription initiation the Shine-Dalgamo sequence and the start codon
AUG (Sambrook, J., Fritsh, E. F., and Maniatis, T., Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Similarly, a
eukaryotic expression vector includes a heterologous or homologous
promoter for RNA polymerase II, a downstream polyadenylation signal, the
start codon AUG, and a termination codon for detachment of the ribosome.
Such vectors can be obtained commercially or assembled by the
sequences described in methods well known in the art, for example, the
methods described below for constructing vectors in general.
EXAMPLES
The present invention is further defined in the following Examples.
It should be understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration only. From
the above discussion and these Examples, one skilled in the art can
ascertain the preferred features of this invention, and without departing
from the spirit and scope thereof, can make various changes and
modification of the invention to adapt it to various uses and conditions.

18
Example 1
This Example illustrates a preferred embodiment wherein cultured
cells were transformed with a GAL4 DNA response element-luciferase
reporter plasmid, a plasmid expressing the coding region of the GAL4
DBD fused to the cDNA coding region for amino acids 2-424 of human p-
catenin (GAL4-p-catenin) and a plasmid expressing full length wild type
human AR (Figure 1). The p-catenin cDNA fragment was made by PCR
amplification of the DNA coding sequence for amino acids 2-424 from
human p-catenin using a pcDNA3.1 p-catenin expression vector
(Invitrogen) as a template and single stranded DNA primers containing
Barn H! and Xba! restriction sites respectively. The amplified DNA
fragment was inserted into the multiple cloning site of the pM plasmid
which was linearized using the restriction enzymes Bam HI and Xba I
(Promega) and which contains the coding sequence for the GAL4 DBD
upstream of the multiple cloning site. The reporter plasmid was made by
sub-cloning five copies of the GAL4 upstream activation sequence (UAS)
into pGL3-Basic (Promega) which contains the cDNA coding sequence for
luciferase. The AR expression vector is human full length AR cDNA in
pcDNA3 (Invitrogen) that was obtained from Leonard Freedman (Sloan
Kettering). The cDNA sequence for human AR is available on the gene
sequence information website for GenBank® (Accession No. M35884,
incorporated herein by reference).
CV-1 cells were cultured on 96 well plates and transfected after 24
hours with the expression and reporter plasmids using the lipofectamine
procedure (Invitrogen). In this procedure, the three different plasmid
constructs used were mixed in cell culture media and lipofectamine and
incubated for 15 minutes according to the manufacturer's instructions
(Invitrogen). The plasmid-lipofectamine mixture was diluted in culture
medium, added to cells, and incubated for 4 hours. The cells were rinsed
and then treated with culture media. Twenty-four hours after transfection,
the cells were treated with androgen agonists and antagonists or vehicle.
After 18 hours, cell lysates were harvested and assayed for luciferase
activity using luciferase reagent (Promega) and a luminometer (Wallac).

19
Other known transfection techniques can be used including, for example,
caicium phosphate precipitation transfection technique.
The plasmid constructs used in the one-hybrid assay are shown in
Figure 1. Dotted lines denote known protein-protein or protein-DNA
interaction regions employed in the one-hybrid assay. Arrows denote
transcription start sites.
Example 2
L929 cells were used to determine if DHT stimulates the interaction
between AR and p-catenin in a cell line that endogenously expresses both
proteins. L929 cells, which express endogenous AR and B-catenin. were
treated with 10 nM DHT, 300 nM hydroxyflutamide (flut), DHT plus flut, or
vehicle (veh) for 17 hours. Cell lysates were harvested, precleared with
protein AJG sepharose, and p-catenin was immunoprecipitated using a
goat polyclonal IgG against p-catenin conjugated to agarose (Santa Cruz
Biotechnology). The immunoprecipitates were analyzed by
polyacrylamide gel electrophoresis followed by Western analysis for AR
and p-catenin (P-cat) as indicated using antibodies from Santa Cruz
(Figure 2). The androgen agonist DHT at 10nM was found to stimulate the
interaction between AR and p-catenin. There was no detectable
interaction between these proteins in the absence of DHT. When cells
were treated with DHT in the presence of a 30-fold excess of the AR
antagonist hydroxyflutamide (300 nM) over DHT, the protein-protein
interaction was attenuated (Figure 2). These results demonstrate that the
AR and p-catenin interaction is stimulated by the androgen agonist DHT.
The results also demonstrate that this interaction is susceptible to
disruption by small molecules such as hydroxyflutamide.
Example 3
Experiments were performed to determine the requirement of each
expression vector for luciferase activity in CV-1 cells.
The assay of the invention is demonstrated to measure the DHT
dependent interaction between AR and p-catenin. The reporter plasmid

20
(GAL4-luciferase) containing the luciferase gene under transcriptional
control of the 5XGAL4-UAS DNA response element was transfected into
CV-1 cells in the presence or absence of the androgen receptor
expression vector (AR), the GAL4-DBD - β-catenin fusion protein
expression vector (GAL4-β-catenin) as indicated. Cells were treated with
1 nM DHT (+) or vehicle (-) for 18 hours where indicated. Cell lysates
were harvested and analyzed for luciferase activity. DHT (1 nM) caused a
large activation of reporter activity when both the AR and GAL4-p-catenin
expression vectors were transfected into the cells with the 5XGAL4-
luciferase reporter (Figure 3). AR and DHT had no effect on reporter
activity in the absence of the GAL4-R-c-atep.in expression vector
demonstrating the absence of a direct effect of AR and DHT on the GAL4
promoter-reporter construct (Figure 3).
Example 4
The estrogen receptor (ER) and progesterone receptor (PR) were
also tested for their ability to interact with p-catenin by measuring their
effect on reporter activity (Figure 4), and the protein-protein interaction
between p-catenin and nuclear receptors was shown to be specific for AR.
CV-1 cells were transfected with the 5XGAL4-luciferase reporter and the
GAL4-p-catenin expression plasmid in the absence of a nuclear receptor
expression vector (-), with the AR, PR, or ER expression plasmid. The
cells were treated for 18 hours with vehicle, 10nm DHT, 10nM
trimegestone (Trim), or 10 nM 17p-estradiol (E2) as indicated. Cell lysates
were harvested and analyzed for luciferase activity. The ER and PR
receptors had no activity in the one-hybrid assay in the presence of
trimegestone, 17p-estradiol, or DHT. There was a large increase in
reporter activity when DHT was added to AR expressing cells but not with
10 nM 17p-estradiol or trimegestone. These results demonstrate that the
interaction of p-catenin in this assay with nuclear receptors appears to be
specific for AR when compared to ER and PR.

21
Example 5
To determine if Applicants' assay has the potential to identify
compounds that disrupt the DHT-dependent interaction of AR and β-
catenin, the effect of cyproterone acetate (CA), an AR antagonist, was
tested in the presence of DHT (Figure 5).
CV-1 cells were transfected with the AR and GAL4-β-catenin
expression and luctferase reporter plasmids described in Figure 1. Cells
were treated for 18 hours with (+) or without (-) 1 nM DHT and the
indicated concentrations of the AR antagonist cyproterone acetate. Cell
lysates were harvested and analyzed for luciferase activity. The DHT
stimulated interaction between AR and R-catenin is inhibited bv the AR
antagonist cyproterone acetate. In the absence of CA, DHT at 1 nM
caused a large activation of reporter activity. This effect of DHT was dose
dependently reversed by CA from between 1 and 1000 nM (Figure 5).
References
1. Yang F, Li X, Sharma M, Sasaki C, Longo D, Lim B, Sun Z. (2002) J.
Biol. Chem. 277:13; 11336-11344.
2. Pawiowski J, Ertel J, Allen M, Xu M, Butler C, Wilson E, Wierman M.
(2002) J. Biol. Chem. 277: 23; 20702-20710.
3. Song L, Herrell R, Byers S, Shah S, Wilson E, Geimann E. (2003) Mol.
Cell. Biol. 23: 5; 1674-1687.
4. Sharma M, Chuang W, Sun Z (2002) J. Biol. Chem. 277: 34; 30935-
30941.
5. Truica C, Byers S, Geimann E. (2000) Cancer Res. 60:4709-4713.
6. U.S. Patent No. 5,283,173, Fields, etal., "System to Detect Protein-
Protein Interactions".
7. U.S. Patent No. 5,468,614, Fields, ef a/., "System to Detect Protein-
Protein Interactions".

22
CLAIMS
What is claimed is:
1. A method of determining if a test compound is able to modulate the
interaction between androgen receptor and p-catenin comprising the steps
of:
(a) providing a cell comprising:
(i) a DNA sequence encoding a hybrid protein
comprising a DNA binding domain fused to the NH3-
terminai region of β-catenin,
(ii) a DNA sequence comprising an upstream activation
sequence corresponding to said DNA binding domain
operably linked to and controlling transcription of a
reporter gene, and
(iii) a DNA sequence encoding androgen receptor protein,
(b) introducing the test compound to the cell, optionally in the
presence of androgen; and
(c) measuring the expression of the reporter gene,
wherein an increase or decrease of expression by the reporter gene
indicates that the test molecule is able to modulate the interaction between
androgen receptor and p-catenin.
2. The method of Claim 1, wherein the DNA binding domain of (i)
comprises a GAL-4 DNA binding domain.
3. The method of Claim 1, wherein the upstream activation sequence
operably linked to a reporter gene of (ii) comprises GAL-4 UAS.
4. The method of Claim 3, wherein there are one or more copies of the
GAL-4 UAS sequence operably linked to the reporter gene.
5. The method of Claim 1, wherein the reporter gene is luciferase.

23
6. The method of Claim 1, wherein the NH3-terminal region of p-catenin of
(i) comprises amino acids 2 through 424 of human p-catenin.
7. The method of Claim 1, wherein the NH3-terminal region of p-catenin of
(i) comprises a nucleotide sequence encoding an amino acid sequence
having at least 65% identity with amino acids 2-424 of SEQ ID NO:1.
8. The method of Claim 7, wherein the NH3-terminal region of p-catenin of
(t) comprises a nucleotide sequence encoding an amino acid sequence
having at least 75% identity with amino acids 2-424 of SEQ ID NO:1.
9. The method of Claim 8, wherein the NH3-terminal region of p-catenin of
(i) comprises a nucleotide sequence encoding an amino acid sequence
having at least 85% identity with amino acids 2-424 of SEQ ID NO:1.

10. The method of Claim 9, wherein the NH3-terminal region of p-catenin
of (i) comprises a nucleotide sequence encoding an amino acid sequence
having at least 95% identity with amino acids 2-424 of SEQ ID NO:1.
11. The method of Claim 1, wherein the NH3-terminal region of P-catenin
of (i) comprises a nucleotide sequence which hybridizes with a nucleotide
sequence encoding amino acids 2-424 of SEQ ID NO:1 under the
following conditions: 6xSSC at 45 °C and washed at least once with
0.2xSSC, 0.1% SDS at 50 °C.
12. The method of Claim 11, wherein the NH3-terminal region of p-catenin
of (i) comprises a nucleotide sequence which hybridizes with a nucleotide
sequence encoding amino acids 2-424 of SEQ ID NO:1 under the
following conditions: 6*SSC at 45 °C and washed at least once with
0.2XSSC, 0.1% SDS at 55 °C.

24
13. The method of Claim 12, wherein the NH3-terminaI region of p-catenin
of (i) comprises a nucleotide sequence which hybridizes with a nucleotide
sequence encoding amino acids 2-424 of SEQ ID N0:1 under the
following conditions: 6xSSC at 45 °C and washed at least once with
0.2*SSC, 0.1% SDS at 65 °C.
14. The method of Claim 1, wherein said cell is a eukaryotic cell.
15. The method of Claim 14, wherein said cell is a mammalian cell.
16. The method of Claim 1, wherein the modulation of expression of the
reporter gene is a decrease in expression.
17. The method of Claim 1, wherein said androgen at step (b) is DHT.
18. The method of Claim 1, wherein the DNA binding domain of (i)
comprises a GAL-4 DNA binding domain; wherein the NH3-terminal region
of p-catenin of (i) comprises amino acids 2 through 424 of human p-
catenin; wherein there are more than one copy of the upstream activation
sequence GAL-4 UAS operably linked to the reporter gene; wherein the
reporter gene is luciferase; wherein the androgen at step (b) is DHT; and
wherein the modulation is a decrease in expression.
19. The method of Claim 1, wherein the NH3-terminal region of p-catenin
comprises armadillo repeats 1-12.
20. The method of Claim 1, wherein the NH3-terminaI region of p-catenin
comprises armadillo repeats 1-7.
21. The method of Claim 1, wherein the NH3-terminal region of p-catenin
comprises armadillo repeats 1-6.

25
22. A method of determining if a test compound is able to modulate the
interaction between androgen receptor and p-catenin comprising the steps
of:
(a) providing a cell comprising:
(i) a DNA sequence encoding a hybrid protein
comprising a DNA binding domain fused to p-catenin,
(ii) a DNA sequence comprising an upstream activation
sequence corresponding to said DNA binding domain
operably linked to and controlling transcription of a
reporter gene, and
(iii) a DNA sequence encoding androgen receptor protein,
(b) introducing the test compound to the cell, optionally in the
presence of androgen; and
(c) measuring the expression of the reporter gene,
wherein an increase or decrease of expression by the reporter gene
indicates that the test molecule is able to modulate the interaction between
androgen receptor and p-catenin.
23. A method of determining if a test compound selectively modulates the
(3-catenin-Wnt signaling pathway over an androgen receptor signaling
pathway comprising:
(a) identifying a test compound which increases or decreases
the expression of a gene by inhibiting the AR mediated
interaction with p-catenin, wherein the test compound
removes androgen-liganded AR repression on Wnt signaling
without repressing androgen-AR mediated transcription; and
(b) assaying the test compound of (a) to determine whether the
test compound increases or decreases the expression of a
gene through a p-catenin independent androgen receptor
signaling pathway;
whereby the test compound of (a) selectively modulates the p-catenin-Wnt
signaling pathway by inhibiting the ability androgen-liganded AR to interact

26
with β-catenin if the test compound fails to increase or decrease the
expression of a gene through an androgen receptor signaling pathway.
24. The method of Claim 23, wherein step (a) comprises the steps of
(A) providing a cell comprising:
(i) . a DNA sequence encoding a hybrid protein
comprising a DNA binding domain fused to p-catenin;
(ii) a DNA sequence comprising an upstream activation
sequence corresponding to said DNA binding domain
operably linked to and controlling transcription of a
reporter gene; and
(iii) a DNA sequence encoding androgen receptor protein;
(B) introducing the test compound to the cell, optionally in the
presence of androgen; and
(C) measuring the expression of the reporter gene,
wherein an increase or decrease of expression by the reporter gene
indicates that the test molecule is able to modulate the interaction between
androgen receptor and P-catenin.
25. The method of Claim 24, wherein the DNA sequence of (i) encodes a
hybrid protein comprising a DNA binding domain fused to the NH3-terminal
region of p-catenin.
26. A method of determining if a test compound selectively modulates an
androgen receptor signaling pathway over a p-catenin-Wnt signaling
pathway comprising:

(a) identifying a test compound which increases or decreases
the expression of a gene through an androgen receptor
signaling pathway; and
(b) assaying the test compound of (a) to determine whether the
test compound increases or decreases the ability of

27
androgen-liganded AR or non-liganded AR to inhibit (3-
catenin/Wnt signaling;
whereby the test compound of (a) selectively modulates an androgen
receptor signaling pathway without removing androgen-liganded AR
repression of Wnt signaling or does not promote the interaction between
AR and p-catenin in the absence of an AR agonist resulting in the test
compound having no activity in increasing or decreasing the expression of
a gene reguiated by p-catenin-Wnt signaling pathway.
27. The method of Claim 26, wherein step (b) comprises the steps of
(A) providing a cell comprising:
(i) a DNA sequence encoding a hybrid protein
comprising a DNA binding domain fused to p-catenin;
(ii) a DNA sequence comprising an upstream activation
sequence corresponding to said DNA binding domain
operably linked to and controlling transcription of a
reporter gene; and
(iv) a DNA sequence encoding androgen receptor protein;
(B) introducing the test compound of (a) to the cell, optionally in
the presence of androgen; and
(C) measuring the expression of the reporter gene,
wherein an increase or decrease of expression by the reporter gene
indicates that the test molecule is able to modulate the interaction between
androgen receptor and p-catenin.
28. The method of Claim 27, wherein the DNA sequence of (i) encodes a
hybrid protein comprising a DNA binding domain fused to the NH3-terminal
region of p-catenin.

Methods for determining if test compounds are able to modulate the interaction between adrogen receptor and beta-
catenin are disclosed. Methods for the determining whether a test compound selectively modulates an adrogen receptor signaling
pathway over a beta-catenin-Wnt signaling pathway or a beta-catenin-Wnt signaling pathway over an androgen receptor signaling
pathway are also disclosed.